Antibodies are large polypeptide complexes (IgG; 150 kDa (˜10 nm)-IgM; 900 kDa (˜40 nm)) used by the immune system to identify and neutralize foreign objects such as bacteria, viruses and toxins in blood or other bodily fluids of vertebrates (FIG. 2c). To neutralize specific antigens in a heterogeneous mixture they recognize specific molecules or fragments of molecules and bind to them with high affinity. Because of their compact size they can diffuse into most parts of the body through blood, tissue fluids and mucus5, 16. Since the first discovery of antibody-mediated immunotherapy over a hundred years ago, antibodies are now widely used as tools in medicine and research1-3. Despite their enormous importance antibodies cannot be employed under all conditions. As proteins their effectiveness can be diminished by extremes in pH, enzymes, temperature and environment (non-aqueous solvents, denaturants)17.
Antibodies are of undisputed importance to research and medicine. Research applications currently include flow cytometry, protein separation and identification, and examining protein expression. When antibodies for a particular epitope, or target protein, aren't available, researchers may need to synthesize their own. There is also a need to produce pure, safe antibodies on a larger scale for use as therapeutics. A new trend is that antibodies are being prescribed for chronic conditions and are given in relatively high, concentrated doses (grams instead of mg) due to their lower potency than other therapeutics. A recent 2007 paper reported that 20% of biopharmaceuticals in clinical trials are monoclonal antibodies, and uses for the diagnosis and treatment of cancer, treatment of rheumatoid arthritis, multiple sclerosis, and psoriasis, prevention of transplant rejection, as use as a prophylactic, have already been approved. In order to meet this demand, purification capacity will need to expand almost 30% each year.
As costs associated with cell culture and harvesting antibodies have decreased, the purification steps, particularly the protein A purification, have come to account for a larger percentage of the expenses associated with antibody production and thus have greater optimization potential. The affinity separation step has been pinpointed by many researchers as a bottleneck in the production process. With current methods, antibodies are produced in cell bioreactors and harvested from cell culture supernatant, then purified via affinity separation before undergoing viral inactivation at low pH, polishing, viral filtration, and the final concentration step. Affinity separation is the primary capture step, and protein A is the most commonly used reagent for the initial antibody capture. These methods are expensive and time consuming.
What is needed are methods for preparing target specific nanoparticles that are stable, less-expensive to manufacture, and that do not rely on harsh conditions such that non-denatured target molecules can be imprinted.